Cytochrome c is a phylogenetically ancient protein and an essential component of the respiratory apparatus of all eukaryotic cells. Although it functions in the mitochondria it is encoded by nuclear genes. In mammals, its biosynthesis involves two independent mechanisms of hormonal and developmental control. Cell-specific levels of a somatic form are present in all tissues of the adult while a testicular variant is expressed only during spermatogenesis. We originally observed a multigene family comprising about 30 nonallelic sequences in the genomes of rat and other mammals. Our molecular analysis during the current grant period has established that most of these arise through the reverse transcription of the multiple heterogeneous mRNA products of a single intron-containing parent gene (RC4) and are dispersed throughout the genome. In addition to RC4, only one such processed gene (RC9) has a nondefective coding sequence and is colinear with all 3 cytochrome c mRNAs (1400, 1100 and 700 nucleotides) and is thus potentially functional. We also observe coordinate transcriptional regulation of the 3 mRNAs in response to thyroid hormone. The itme course of mRNA induction parallels that for the enhanced respiratory activity mediated by these hormones. Our specific objectives for this proposed project period are as follows: 1) To continue our studies of cytochrome c gene structure and expression with particular emphasis on the characterization of genes and pseudogenes encoding the testi-specific molecular variant of cytochrome c and its related transcription products. The differential pattern of gene expression for both testicular and somatic forms will be defined for the various stages of spermatogenic differentiation. 2) To evaluate and apply various gene transfection techniques to the analysis of cis-acting sequences required for cytochrome c gene function and its hormonal control as well as to the putative role of repetitive DNA elements in promoting the activity of RC9. Transcriptionslly active regions of regulatory significance will be defined through the transfection of mutant genes constructed in vitro into biological systems which display physiologically relevant responses to hormonal stimulation. 3) To utilize yeast as a recipient host organism for the direct in vivo analysis of mutations associated with the numerous processed cytochrome c pseudogenes in mammalian genomes. The focus will be on defining parameters for efficient heterologous expression and the characterization of mutations in invariant but functionally undefined regions of the polypeptide.